According to the estimate made by World Health Organization (WHO), nearly one-third of the world populations are infected with tuberculosis (TB) and around eight million new cases were reported every year. In Taiwan, registered new tuberculosis cases have increased dramatically in the past few years, and approximately sixty out of a hundred thousand people were infected at present. However, only three-forth of the patients were receiving the treatments. As indicated by the Department of Health (DOH), 4.2 people died of tuberculosis everyday in Taiwan, and hepatotoxicity and neurological damage, e.g. auditory and optic neuroninjury, etc. are common clinical side effects observed in patients treated with TB drugs. Among which, hepatotoxicity is the most commonside effect reported. Furthermore, due to the fact that chronic hepatitis B and C are prevailing diseases in Taiwan, if 14,000 people were infected with tuberculosis each year, it is estimated that approximately 2,000 to 3,000 people among those active tuberculosis patients also have chronic liver disorders and require the treatment of tuberculosis. Therefore, the most universal side effect of the tuberculosis treatment, hepatotoxicity, is an iatrogenicdisorder that should not be neglected.
Most primary anti-tuberculosis drugs, e.g. isoniazid, pyrazinamide, and rifampin, have potential side effects such as hepatotoxicity. Among those drugs, isoniazid is the most effective, but also the one that can easily induce hepatotoxicity. Isoniazid induced hepatotoxicity has been reported since the late 60's, and roughly 0.1 to 1% of the treated patients showed clinical symptoms of hepatotoxicity (Kopanoff D E et al., Isoniazid-related hepatitis: a U.S. Public Health Service cooperative surveillance study, 1978. Am. Rev Respir Dis 117:991-1001; Nolan C M et al., Hepatotoxicity associated with isoniazid preventive therapy: a 7-year survey from a public health tuberculosis clinic. 1999. JAMA 281: 1014). Moreover, 10 to 20% of those patients exhibited abnormal liver functions in the absence of clinical symptoms, and the first sign of liver malfunction usually took place two months after the initial treatment of isoniazid (Steele M A et al., Toxic hepatitis with isoniazid and rifampin: A meta-analysis. 1991. Chest. 99: 465).
As shown in FIG. 1, the major pathway of isoniazid metabolism is acetylation to acetylisoniazidby N-acetyltransferase (NAT) followed by rapidly hydrolysis to isonicotinic acid and acetylhydrazine. Acetylhydrazine can be further acetylated into either non-toxic diacetylhydrazine or toxic molecules which include acetyldiazene, acetylonium ion, acetylradical, and ketene etc. by N-acetyltransferase and Cytochrome P450 2E1 (CYP 450 2E1), respectively. Additionally, in the presence of oxygen and NADPH, acetylhydrazine can react with Cytochrome P450 2E1 and produce free radicals, and such oxidation stress can induce cell death. Moreover, both isoniazid and acetylhydrazine can be hydrolyzed to toxic hydrazine by amidase.
Recent studies have indicated that hydrazine (not isoniazid or acetylhydrazine) is most likely to be responsible for INH-induced hepatotoxicity observed in rabbits and rats, and the severity of hepatotoxicity is positively correlate with the concentration of hydrazine (Sarich T C, Youssefi M, Zhou T, Adams S P, Wall R A, Wright J M. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835-840; Yue J, Peng R X, Yang J, Kong R, Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704.). Sarich et al. in 1999 reported that bis-p-nitrophenyl phosphate (BNPP), an inhibitor of amidase, can prevent isoniazid-induced hepatotoxicity by inhibition of hydrazine production (Sarich T C, Adams S P, Petricca G, Wright J M Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor. 1999. J Pharmacol Exp Ther. 289: 695-702).
Cytochrome P450 2E1 (CYP2E1) is constitutively expressed in liver and is involved in metabolic pathways of many compounds, e.g. CCl4 and acetaminophen (Lee S S, Buters J T, Pineau T, Fernandez-Salguero P, Gonzalez F J. Role of CYP2E1 in the hepatotoxicity of acetaminophen. 1996. J Biol Chem 271: 12063-12067; Wong F W, Chan W Y, Lee S S. Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. 1998. Toxicol Appl Pharmacol. 153: 109-118). Nevertheless, the role of CYP22E1 in isoniazid-induced hepatotoxicity remains unclear. Isoniazid is an inducer of CYP22E1 (Ramaiah S K, Apte U, Mehendale H M. Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29: 1088-1095.). Some studies have suggested that CYP22E1 in liver is involved in the mechanism of isoniazid-induced hepatotoxicity (Yue J, Peng R X, Yang J, Kong R, Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704; Huang Y S, Chem H D, Su W J, Wu J C, Chang S C, Chiang C H, Chang F Y, et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. 2003. Hepatology 37: 924-930.). In vitro studies have also suggested that disulfuram (DSF) and its metabolite, diethyldithiocarbamate, are the selective mechanism-based inhibitors for CYP2E1 in human liver microsomes (Guengerich F P, Kim D H, Iwasaki M. Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. 1991. Chem Res Toxicol. 4: 168-179; Hunter A L, Neal R A Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205.). Brady et al. have demonstrated that oral administration of a single dose of disulfuram (DSF) in rats can result in immunoreactive hepatic content and rapidly reduce the activity of CYP2E1 (Brady J F, Xiao F, Wang M H, Li Y, Ning S M, Gapac J M, Yang C S. Effects of disulfuram on hepatic P45011E1, other microsomal enzymes, and hepatotoxicity in rats. 1991. Toxicol Appl Pharmacol. 108: 366-373.).
Sodhi et al. reported in 1997 that oxidative-stress is one of the factors that contribute to the hepatotoxicity induced by isoniazid and rifampicin in young rats (Sodhi C P, Rana S V, Mehta S K, Vaiphei K, Attari S, Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. 1997. Drug Chem Toxicol 20: 255-269). Numerous research focused on identification of appropriate biomarkers so as to evaluate the in vivo rate of oxidation has discovered three types of biomarkers: biomarkers for damage caused by lipid, protein and nucleic acid oxidation. 8-iso-prostaglandin F2α (8-iso-PGF2α) is the product of lipid oxidation of arachidonic acid and is chemically stable. The amount of 8-iso-PGF2α can be used as an indicator for in vivo lipid oxidation and the oxidation is likely related to the production of free radicals, oxidative damage, and antioxidant deficiency (Morrow J D, Hill K E, Burk R F, Nammour T M, Badr K F, Roberts L I, 2nd. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. 1990. Proc. Natl. Acad. Sci. USA 87: 9383-9387; Morrow J D. The isoprostanes: their quantification as an index of oxidant stress status in vivo. 2000. Drug Metab Rev. 32: 377-385.). Presently, many methods are available for measuring the concentration of 8-iso-PGF2α which include enzyme immunoassay (Devaraj S, Hirany S V, Burk R F, Jialal I. Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes. 2001. Clin. Chem. 47: 1974-1979.); radioimmunoassay (Helmersson J, Basu S. F2-isoprostane excretion rate and diurnal variation in human urine. 1999. Prostaglandins Leukot. Essent. Fatty Acids 61: 203-205.); gas-chromatography mass spectrometry (Morrow J D, Roberts L J, 2nd. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. 1999. Methods Enzymol. 300: 3-12.) and liquid chromatography mass spectrometry (Li H, Lawson J A, Reilly M, Adiyaman M, Hwang S W, Rokach J, FitzGerald G A. Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F(2)-isoprostanes in human urine. 1999. Proc. Natl. Acad. Sci. USA 96: 13381-13386.) etc. In addition, 8-iso-PGF2α in human urine and its metabolite, 2,3-dinor-8-iso-PGF2α, can be extracted by C18 solid phase extraction (SPE) and then apply to LC/MS/MS analysis (Liang Y, Wei P, Duke R W, Reaven P D, Harman S M, Cutler R G, Heward C B. Quantification of 8-iso-prostaglandin-F2α and 2,3-dinor-8-iso-prostaglandin-F2α in human urine using liquid chromatography-tandem mass spectrometry. 2003. Free Radic. Biol. Med 34: 409-418.).
Currently, the available tests for assessing liver function so as to monitor the progress of liver damage and screen for chronic liver diseases include both conventional and quantitative tests. The most common tests used are examining the concentrations of plasma aspartate aminotransferase (AST), plasma alanine aminotransferase (ALT), plasma alkaline phosphatase, and liver metabolites, e.g. bilirubin and albuminetc.; or studying the coagulation factorsby measuring the prothrombin time etc. (Carlisle R, Galambos J T, Warren W D. The relationship between conventional liver tests, quantitative function tests, and histopathology in cirrhosis. 1979. Dig. Dis. Sci. 24: 358-362.).
The tests of liver function mostly are based on the turn-over or time-dependent serum concentrations of a test substrate that is metabolized almost exclusively via the liver (hepatic elimination). The clearance of such substrates is determined by the hepatic portal vein and hepatic artery blood flow, as well as by the extraction of these substances by the liver. The hepatic blood flow correlates with the amount of the substances supplied to the liver. On the other hand, its elimination is determined by the hepatic metabolic capacity (Herold C, Heinz R, Niedobitek G, Schneider T, Hahn E G, Schuppan D. Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. 2001. Liver 21: 260-265.).
Galactose is one type of carbohydrates that has high extraction ratio and 90% of its metabolism was processed in liver. In Liver, galactose was epimerized to glucose-1-phosphate by galactokinase and the reaction of galactokinase is the rate-limiting step in galactose metabolism. Due to the high extraction ratio of galatose and related hepatic blood flow, galactose elimination capacity became the most widespread test for examining liver function. At present, no specific test was available for evaluating residual liver function in rats, hence, measuring the metabolism capacity of a definite compound (e.g. galactose) can provide information on both rate-limiting step(s) in liver metabolism and representative value of residual liver function (Keiding S, Johansen S, Tonnesen K. Kinetics of ethanol inhibition of galactose elimination in perfused pig liver. 1977. Scand J. Clin. Lab Invest. 37: 487-494; Keiding S, Johansen S, Winkler K. Hepatic galactose elimination kinetics in the intact pig. 1982. Scand J. Clin. Lab Invest. 42: 253-259).
Galactose elimination capacity (GEC) is a well-established quantitative test for assessing human liver function (Lindskov J. The quantitative liver functions as measured by the galactose elimination capacity. I. Diagnostic value and relations to clinical, biochemical, and histological findings in patients with steatosis and patients with cirrhosis. 1982. Acta Med. Scand. 212: 295-302). Nonetheless, the requirement of obtaining multiple blood samples so as to establish a standard curve impedes its clinical applications. Consequently, galactose single point (GSP) test was used instead in numerous studies to assess human liver function. The inventor(s) of the present invention used GSP method to test liver function of patients with chronic hepatitis; liver cirrhosis; and hepatoma, and demonstrated that GSP test can precisely identify these liver disorders (Tang H S, Hu O Y. Assessment of liver function using a novel galactose single point method. 1992. Digestion 52: 222-231). Moreover, previous study has shown that GSP test can be successfully applied to measuring the residual liver function among patients with chronic liver diseases after treatment of promazine and cefoperazone (Hu O Y, Tang H S, Chang C L. The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone—a novel galactose single-point method as a measure of residual liver function. 1994. Biopharm Drug Dispos 15: 563-576; Hu O Y, Hu T M, Tang H S. Determination of galactose in human blood by high-performance liquid chromatography: comparison with an enzymatic method and application to the pharmacokinetic study of galactose in patients with liver dysfunction. 1995. J. Pharm. Sci. 84: 231-235; Hu O Y, Tang H S, Sheeng T Y, Chen T C, Curry S H. Pharmacokinetics of promazine in patients with hepatic cirrhosis—correlation with a novel galactose single point method. 1995. J. Pharm. Sci. 84: 111-114). In addition, GSP test was recommended by FDA, U.S.A. in the published “Guidance for Industry” to be used as one of the tests for assessing liver function (FDA Center for Drug Evaluation and Research (CDER) Pharmacokinetics in patients with impaired hepatic function: Study design, data analysis, and impact on dosing and labeling. Guidance for Industry, U.S. Department of Health and Human Service. 2003. pp 5). In conclusion, the primary anti-tuberculosis drug, isoniazid, has many side effects and is not well-designed, hence, improvement is much needed.